JPH02223553A - Novel antibiotic rk-441, production thereof, antitumor agent and immunosuppressive agent - Google Patents

Abstract

NEW MATERIAL:An antibiotic substance RK-441 expressed by the formula. USE:An antitumor agent and immunosuppressive agent. PREPARATION:A microorganism [e.g. Streptomyces espi RK-441 (FERM P-10306)] belonging to the genus Streptomyces having producing ability of antibiotic substance RK-441 is inoculated into culture medium and cultured by a method such as submerged culture to provide the antibiotic substance RK-441 expressed by the formula. Nutrition source of the culture medium is not especially limited and can contain ordinary carbon source (e.g. starch, dextrin or glycerin), nitrogen source (e.g. peptone, soybean powder, etc.). The pH of the culture medium is preferably 4-9, especially preferably nearly neutral and temperature thereof is preferably about 25-35 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a novel antibiotic RK-441, a method for producing the same, and antitumor agents and immunosuppressants containing RK-441 as an active ingredient.

[Background of the invention]

In recent years, the causes of disorderly proliferation of cancer cells are being elucidated at the molecular level. It is assumed that cells become cancerous when the expression of oncogenes (oncogenes) increases or when growth factor signal transmission becomes abnormal.

Many of the conventional anticancer antibiotics are
Recently, there has been a strong desire for the development of anticancer drugs based on new mechanisms of action, such as inhibition of oncogene expression and inhibition of growth factor transmission.

Furthermore, the development of immunosuppressants useful for the treatment and prevention of transplant rejection, graft-versus-host disease caused by bone marrow transplantation, autoimmune diseases, infectious diseases, etc. is actively underway, and new immunosuppressants are being developed. If it does, the effect will be huge.

[Purpose of the invention]

An object of the present invention is to provide a novel antibiotic having antitumor activity and immunosuppressive action and a method for producing the same. Furthermore,
An object of the present invention is to provide an antitumor agent and an immunosuppressant containing the above antibiotic as an active ingredient.

[Structure of the invention]

The antibiotic RK-441 of the present invention is a novel antibiotic represented by the following formula and which has not been described in any literature. Antibiotic RK-44
Since compound No. 1 exhibits an excellent suppressive effect on tumor cells, has an immunosuppressive effect, and is not cytotoxic, it can provide an extremely safe antitumor agent and immunosuppressant.

The present invention will be explained in detail below.

Production of Antibiotic RK-441> (Microorganisms Used) The microorganisms that produce the antibiotic RK-441 of the present invention belong to the genus Streptomyces and have the ability to produce the antibiotic RK-441.

As an example, Streptomyces sp. RK-44
1 (Streptomyces sp, RK-441
) (hereinafter referred to as "RK-441 strain"), which has the above-mentioned characteristics and advantageously produces the antibiotic RK-441 of the present invention. It can be effectively used in the production method.

In addition, not only natural and artificial mutant strains of the above-mentioned RK-441 strain, but also all microorganisms belonging to the genus Streptomyces that have the ability to produce antibiotic R-441, which will be described later, may be used in the method of the present invention. Can be done.

The above R. Niichi 441 strain is a soil actinomycete isolated from soil collected in Nirasaki City, Yamanashi Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on September 30, 1986.
The microorganism accession number is FIKEN Microbiology No. 1.0306 (
FERM P-10306).

The RK-441 strain has the following mycological properties.

1. Morphological characteristics This strain, RK-441 strain, was cultured in a liquid medium containing 1% yeast extract and 1% glucose, and the cells were cultured with 6N hydrochloric acid and 1% glucose.
LL-diaminopyridic acid was detected in thin layer chromatography of the product hydrolyzed at 10°C for 18 hours, but
Mesodiaminopimelic acid was not detected. When observed under an electron microscope when grown on an agar plate, the aerial mycelia exhibit an incomplete spiral shape, and the spore surface is smooth and cylindrical.

2 Growth conditions on various media (cultured at 27°C for 20 days, color tone is based on Descriptive Color Names Dictionary)
(according to Ames Dictionary) 1) Starch yeast agar medium Growth: Normal aerial hyphae: Normal aerial hyphae Color tone: 4 ig (light yellowish brown) Back side color tone = 7m
p (chocolate color) Soluble pigment: 5p. (Chocolate brown) 2) Yeast extract/malt extract agar medium Growth: Normal aerial mycelium: Normal aerial mycelium Color tone: 4 ig (light yellow brown) Back side color tone, 7 companies (chocolate color) Soluble pigment: Dark wine color 3) Oat mill Growth on agar medium: No mycelium Back color: 3 cd (ocher) Soluble pigment: None 4) Growth on starch/inorganic salt agar medium: No mycelium Back side color: 3 cd (ocher) Soluble pigment : None 5) Growth on tyrosine agar medium Normal aerial mycelium, None Back side color: 4j2g (yellow brown) Soluble pigment: Light brown 6) Sucrose nitrate agar medium Growth: Poor aerial mycelium: None Back side color tone: 3cd (loess) Color) Soluble pigment: None 7) Growth on glucose/asparagine agar medium: None Underside color tone: 3ie (camel color) Soluble pigment: Dark red 6) Growth on criceol/asparagine agar medium: None Back side color tone: 4ng (maple color) Soluble pigment: 6ie (red and sod color) 9) Nutrient agar medium growth: Normal aerial mycelia: None Back side color tone: 4il (light brown) Soluble pigment: None 10) Peptone yeast extract・Growth on iron agar medium:
Unfavorable mycelium: None Back color: 4ie (yellow brown) Soluble pigment: None 3 Sugar utilization D-Glucose D-Fructose D-Xylose L-Rhamnose Raffinose L-Arabinose Good growth Good growth Good growth Normal growth Normal growth Sucrose: No growth Inositol: No growth From the above characteristics, it is clear that the RK-441 strain belongs to the genus Streptomyces. However, Bergey's Manual of Determinative Bacteriology
Manual of Determinative
Bacteriology) and International
International Journal of Systematic Bacteriology
Systematic Bacteriology)
It did not match any of the bacterial species described. Streptomyces pulveraceus has similar properties.
), but this bacterium can be grown on oatmeal agar medium,
Aerial mycelium formation was observed in starch mineral salt medium, and RK-44
The difference is that a single strain cannot produce aerial mycelium.

From the above points, RK-44] strain is Streptomyces pulveraceus (Streptomyces pulver
It is thought to be a new bacterial species closely related to P. aceus).

(Culture method and purification method) In order to obtain the antibiotic RK-441 of the present invention, the above-mentioned antibiotic-producing bacteria belonging to the genus Streptomyces may be cultured by an unusual method for producing antibiotics. The form of culture may be liquid culture or solid culture, and for industrially advantageous culturing, a spore suspension or culture solution of the above-mentioned producing bacteria may be inoculated into a medium and culture with aeration and stirring may be performed.

The nutrient source of the medium is not particularly limited, and the medium may contain carbon sources, nitrogen sources, and other sources commonly used for culturing microorganisms. Carbon sources include starch,
Dex), glycerin, glucose, sucrose, galactose, inositol, mannitol, etc. Nitrogen sources include peptone, soybean flour, meat extract, rice bran, wheat gluten, urea, cornstarch liquor, ammonium salts, nitrates, Other organic or inorganic nitrogen compounds may be used. Other inorganic salts, such as table salt,
Phosphates, metal salts such as potassium, calcium, zinc, manganese, iron, etc. may be added as appropriate, and animal, vegetable, mineral oils, etc. may be added as antifoaming agents, if necessary. Culture conditions such as culture temperature and culture time are suitable for the growth of the bacteria used.
Furthermore, conditions are selected to maximize the production of RK441.

For example, the pH of the medium should be between 4 and 9, especially around neutrality.
The appropriate culture temperature is preferably about 25 to 35°C. However, these culture conditions such as culture composition, hydrogen ion concentration of the medium, culture temperature, and stirring conditions should be adjusted as appropriate to obtain favorable results depending on the type of bacterial strain used and external conditions. Needless to say, it is. In order to obtain RK-441 from the culture thus obtained, any means commonly used for collecting metabolites can be used as appropriate. For example, any of the methods that utilize the solubility difference between RK-441 and impurities, the ionic binding force difference, the adsorption affinity difference, and the molecular weight difference may be used alone or , used in appropriate combinations or repeatedly. Specifically, most of RK-441 is present in the culture filtrate. When the culture solution is purified using a combination of various ion exchange chromatography, gel filtration chromatography, adsorption chromatography, liquid chromatography, cellulose partition chromatography, etc., both components containing RK-441 and other active components can be obtained. . The powder obtained by freeze-drying this fraction is further purified by high performance liquid chromatography (e.g., developed with Nucleozil, CO8, 50% methanol system) to obtain purified white powder of RK-441.

The physicochemical properties and biological properties of RK-441 thus obtained are as follows.

Physicochemical and biological properties of antibiotic RK-441> Melting point: 185-187°C Elemental analysis: C61,39H6,52N9.41% Specific optical rotation: [α]. -15.3° (co, 22, MeOH
) Ultraviolet absorption spectrum: Shown in Figure 1.

Infrared absorption spectrum: Shown in Figure 2.

3400.3199.3093 2976.2940.2905. 2874.2361.1725. 1701.1676.1643. 1584.1458.1386. 1332.1293.1269. 1230, 1200.1139. 1094.1048.1028. 931.873.813. 787.650.641. 625.582.459. 442 cm-' Solubility: Easily soluble in chloroform and methanol, soluble in water,
Insoluble in hexane Molecular weight: 292.1423 (based on high-resolution mass spectrometry) (Molecular formula: C, 5H2, N20.) Color reaction: Lydon-Smith, Anthrone, positive for anisaldehyde reaction BCG, ninhydrin, remubenzidine, 2 .4-dinitrophenylhydrazine, color of negative substance for FeCfls reaction: White powder thin layer chromatography m: Thin layer plate manufactured by Merck, USA 0.25 mm Silica gel 60F254 Solvent Chloroform; Methanol = 4: IRf value 0.
87 Solvent Chloroform: Methanol-9; IR [value 0.5
5. Antibacterial spectrum: Shown using the conventional disk assay method on agar plates.

<Comparison with other substances> Antibiotics with these physicochemical and biological properties can be represented by the structural formula below.

It was concluded that RK-441 is a new antibiotic, as it has not been described in the literature.

(Anti-tumor agent containing RK-441 as an active ingredient) R of the present invention
Antitumor agents containing K-441 as an active ingredient can be administered either orally or parenterally, and when administered orally, they can be administered as soft or hard capsules, tablets, granules, fine granules, or powders. When administered parenterally, subcutaneous or intravenous injections such as aqueous suspensions and oil-based preparations, infusions, and solid or viscous suspensions may be used to maintain sustained mucosal absorption, such as herbal medicines. It can be administered in various dosage forms.

The formulation of the active ingredient of the present invention includes surfactants, excipients, lubricants, adjuvants, and pharmaceutically acceptable film-forming substances in order to form an enteric formulation in consideration of the properties of the active ingredient. This can be carried out as appropriate using a coating aid or the like, and specific examples thereof are as follows.

In order to improve the disintegration and elution of the composition of the present invention,
One or more surfactants such as alcohols, esters, polyethylene glycol derivatives, fatty acid esters of sorbitan, sulfated fatty alcohols, etc. can be added.

In addition, as excipients, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, light silicic anhydride, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, Carboxymethyl cellulose calcium and the like can be added alone or in combination of two or more.

As lubricants, for example, one or more types of magnesium stearate, talc, hydrogenated oil, etc. can be added, and as flavoring and flavoring agents, salt, saccharin, sugar, mannitol, orange oil, licorice, etc. can be added. Extracts, sweeteners such as citric acid, glucose, menthol, eucalyptus oil, and malic acid, fragrances, colorants, preservatives, and the like may be included.

Adjuvants such as suspensions and wetting agents include, for example, coconut oil, olive oil, sesame oil, peanut oil, calcium lactate,
Safflower oil, soybean phospholipids, etc. can be contained.

In addition, film-forming substances include cellulose and cellulose acetate phthalate (CAP), which is a carbohydrate derivative such as sugars.
In addition, examples of polyvinyl derivatives such as acrylic acid copolymers and dibasic acid monoesters include methyl acrylate/methacrylic acid copolymers and methyl methacrylate/methacrylic acid copolymers.

In addition, when coating the above-mentioned film-forming substances, in addition to commonly used coating aids such as plasticizers, the properties of the film-forming agent can be improved by adding various additives to prevent the chemicals from adhering to each other during coating operations. This can improve the coating process and make coating operations easier. Note that a dosage form may be prepared in which the active ingredient is microencapsulated using a film-forming substance and then mixed with excipients and the like.

In particular, the blending ratio in typical dosage forms is as follows.

Active ingredient Excipient Lubricant Surfactant Film-forming substance 0.1-90 wt% 10-99.8 〃 0-50 〃 0-50 /7 0.1-50 Particularly preferred range 01-15 wt% 85 ~99.4 0-20 0-20 0.3-20 Particularly preferred excipients are lactose, crystalline cellulose, and calcium carboxymethyl cellulose.

In addition, the dosage is sufficient to effectively treat the target tumor, and depends on the symptoms of the tumor, route of administration, dosage form, etc., but in general, when administered orally, adults receive approximately 0.01-5 mg/kg body weight (about 0 for dwarfs)
．． In the range of 0.1 to 3 mg/kg body weight), the first limit is preferably about 2.5 mg/kg body weight, more preferably about 0.5 mg/kg body weight, and in the case of parenteral administration, the The upper limit is about 0.5 mg/kg body weight, preferably about 0.25 mg/kg body weight, more preferably about 0.5 mg/kg body weight. ], mg/kg body weight is appropriate.

The present invention will be specifically explained below using production examples, formulation examples, and test examples, but the present invention is not limited thereto.

In addition, "%" represents weight %.

Production example Glucose 2%, soluble starch 1%, meat extract 0.1
%, dry yeast 04%, soybean flour 2.5%, salt 02%, dibasic potassium phosphate 0.005% 72f!
The above RK-44I strain was inoculated into a medium of
Cultured with shaking for hours. This culture solution was adjusted to pl (3 pl) with hydrochloric acid and filtered to obtain a filtrate of 60 p, so the active substance was extracted with ethyl acetate. 27.7 g of the oily active substance obtained from the ethyl acetate layer was transferred to a silica gel column. (7,4
X 40 cm) and developed with an eluent of chloroform:methanol-96.4. After concentrating and drying the active fraction (3.5 g), dissolve it in a small amount of chloroform,
Loaded on top of a silica gel column (24 x 56 cm). This was developed with chloroform:methanol-92:8 to obtain 107 mg of active substance. When the active substance dissolved in 80% methanol was added to the top of a Sephadex LH20 column (2.6×95 cm) and developed with 80% methanol, the active substance was eluted with a volume of 400 rd. After concentrating this and lyophilizing it, 34mg
of powder was obtained. Finally, 9.2 mg of pure RK441 substance was obtained by high performance liquid chromatography. The column used was Nuku 1 Noozil sc+o (20x 300m
m), the solvent is 50% methanol and the flow rate is 7.0.
It was set to min.

Formulation Example 1 (Injection/Drop) 5 g of powdered glucose was added to contain 10 mg of RK-441, dispensed aseptically into vials, and sealed.
Store in a cool, dark place sealed with an inert gas such as nitrogen or helium. Before use, add 100 g of 0.85% physiological saline to make an intravenous injection, and administer 10 to IOM per day by intravenous injection or drip depending on the symptoms.

Formulation Example 2 (Injection/Drop) Using 2 mg of RK-441, make an intravenous injection for mild symptoms by the same method as Formulation Example 1, and administer 10 to 100 doses per day depending on the symptoms by intravenous injection or Administer by intravenous drip.

Formulation Example 3 (Enteric-coated capsules) After thoroughly mixing 0.5 mg of RK-441, 2.46 g of lactose, and 0.04 g of hydroxypropyl cellulose, it was formed into granules according to a conventional method, and the mixture was thoroughly dried. The granules are then sieved to produce granules suitable for bottles, heat-seal packaging, etc. Next, 05g of cellulose acetate phthalate
and hydroxypropyl methylcellulose phthalate 0
．． 5 g is dissolved to form a coated base material, and the base material is coated while the granules are allowed to float and flow, thereby producing enteric-coated granules.

Enteric-coated capsule preparation 1 by filling this composition into capsules
Manufacture 00 pieces.

Test Example 1 (DNA synthesis inhibition test of mouse epithelial cells generated in response to epidermal growth factor (EGF)) EGF (5 ng/mn
) and RK-441 and 17 hours later, 3H-labeled thymidine was added to the medium (IμCi/me). 5
The amount of DNA synthesis was determined by counting the radioactivity of the time-labeled acid-insoluble fraction of the cells using a liquid scintillation counter. The inhibition rate was calculated by the following method.

Inhibition rate (%) The results are shown below.

Test compound concentration (μg7ml Inhibition rate (%) RKi4
1 0.3 99.9〃0.1
99.9 〃0.03 99.9 〃0.01 991 〃-0,003765 Cytotoxicity No No No No No No Test Example 2 (Effect on rat cells (NRK) transformed with temperature-sensitive oncogene (srctS)) srctS
- When NRK cells are cultured at 32°C, spherical cells increase due to transformation, but when cultured at 39°C, spherical cells disappear and only flat, adherent cells remain. The drug was added to cells cultured at 32°C, and 24 hours later, the cells were observed under a microscope and spherical cells were counted.

The evaluation of drug efficacy was calculated as follows.

Suppression rate (%) The results are shown below.

Test compound Concentration (μgzW) Inhibition rate (%) Cytotoxic RK-4410,395 None 0.1 95 None 0.03
95 None〃0.01 80
None 0.003 65 None Test Example 3 (Test for suppressing juvenileization of mouse pancreatic cells in response to concanavalin A (ConA)) Balb/c mouse pancreatic cells were prepared, and 10μ
g/ml ConA was added. After culturing the cells for 2 days in a carbon dioxide incubator, MTT reagent (Dimethylt
hiazoyl diphenyl tetrazol
ium bromide) was added, and the increase in absorbance at 570 nm was measured.

Since the increase in absorbance at 570 nm correlates with the juvenileization of lymphocytes, it can be seen that the addition of the RK-441 substance increases the absorbance and suppresses juvenileization.

The inhibition rate was calculated by the following method.

Inhibition rate (%) The results are shown below.

Test compound Concentration (nM) RK-44137 〃12.3 〃4,1 〃1,4 〃0.46 〃0.15 (Effect of the invention) From the above test results, the antibiotic of the present invention has the following inhibition rate: (%) 84.0 62.2 42.9 38.4 25.1 Cytotoxicity None None None None Shows high tumor and immunosuppressive effects on cells at extremely low concentrations. Furthermore, no cytotoxicity was observed at that concentration, making it a highly safe drug.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the ultraviolet absorption spectrum of the antibiotic RK-441 of the present invention, and FIG. 2 is a diagram showing the infrared absorption spectrum (in KBr) of the antibiotic RK-441. Written amendment (method) 1. Indication of the case 1999 Patent Application No. 1.35348 3. Person making the amendment Name of applicant related to the case RIKEN 4゜Representative 5. Date of amendment order 1999 September 26, 2015 6. Documents and drawings certifying the authority of representation subject to amendment (Figure 2)

Claims (5)

[Claims] (1) Antibiotic RK-441 represented by the following formula. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (2) Streptomyces
A method for producing antibiotic RK-441, which comprises culturing antibiotic RK-441-producing bacteria belonging to the genus and separating and collecting antibiotic RK-441 from the culture. (3) The antibiotic RK-441 producing bacteria is Streptomyces sp.
sp. RK-441) The manufacturing method according to claim (2). (4) An antitumor agent containing the antibiotic RK-441 as an active ingredient. (5) An immunosuppressant characterized by containing antibiotic RK-441 as an active ingredient.